Here, we investigate DEK-derived peptides that contain the SAF-box motif. We show that the SAF box alone (amino acids 137–187) (Figure 1B) appearsnot to interact with DNA in solution. However, when many SAF boxes are brought into close proximity, cooperative effects lead to high affinity for DNA. A peptide with amino acids 87–187 (including a sequence of ∼50 amino acids on the N-terminal side of the SAF box) binds to DNA much like the intact DEK preferring four-way junction DNA over straight DNA. This peptide formslargeaggregates in the presence of DNA and is also able to introduce supercoils into relaxed circular DNA. Interestingly, however, the 87–187 amino acid peptide induces negative rather than positive supercoils (as the full-length DEK does). Relatively shortstretches of adjacent amino acid sequences are required for the introduction of positive supercoils.

For protein expression, the vectors were transformed in E.coli BL2A (DE3) Lys S. The purification of the his-tagged DEK fragments was performed by Ni-NTAAgarose according to the manufacturer's protocol (Qiagen) and as described previously (15).

Pull down DNA-binding assays

Aliquots containing 10 μl of settled Protein-A Sepharose beads were incubated with 6 μg of monospecific polyclonal DEK antibodies for 1 h at 4°C in nE450 buffer (20 mM HEPES, pH 7.6, 450 mM NaCl, 10 mM sodiumbisulfite and 1 mM EDTA) in the presence of 1 μg/μl BSA (NewEngland Biolabs) + 0.5% NP40. The beads were washedtwice with 1 ml nE450. The fragments 87–138 and 137–187 were dialyzed against nE300 buffer (20 mM HEPES, pH 7.6, 300 mM NaCl, 10 mM sodium bisulfite, 1 mM EDTA and 1 μg/μl BSA) overnight and incubated at 200 ng polypeptide/200 μl nE450 for 1 h at 4°C with the coupled Protein-A Sepharose beads. The beads were then washed threetimes with nE100 buffer (20 mM HEPES, pH 7.6, 100 mM NaCl, 10 mM sodium bisulfite and 1 mM EDTA) and 30 ng of radiolabeled and double-digested pMII plasmid (17,18) were added. Competition was carriedout with unlabeled, MspI-digested pBlueBacHis2A-vector DNA (fragments rangingbetween 50 and 1500 bp). After 1 h incubation at 4°C, the beads were washed three times with nE100 and then eluted three times with 100 μl 2% SDS. The eluates were pooled and precipitated by the Wessel–Flügge method (19). The first supernatant was precipitated with 3 M sodium acetate for DNA analysis. The DNA was separated on a 0.6% agarose gel in 0.5× TBE at 2 V/cm and analyzed by autoradiography. Proteins were loaded on an 18% SDS–PAGE and analyzed by immunoblotting.

For the investigation of the full-length DEK, a DNA precipitation assay was used. An aliquot of 600 ng of recombinant DEK (14,15) was incubated in a total volume of 30 μl buffer P (10 mM Tris–HCl, pH 8, 80 mM NaCl and 1 mM MgCl2) in the presence of radiolabeled DNA and competitor DNA for 15 min at roomtemperature. The resulting protein–DNA aggregates were centrifuged for 15 min at 14 000 r.p.m. and the supernatant was collected. The pellets were resolved in 2% SDS and bothfractions were analyzed on an agarose gel as described above. For the investigation of a preferential binding of fragments 1–187 and 68–226 to different topological DNA forms, we used the DNA precipitation assay as just described. DNA substrate was partially relaxed SV40 DNA. The assay was performed in a total volume of 30 μl buffer P with 20 ng of DNA and increasing amounts (12.5, 25, 50, 100 and 200 ng) of dialyzed (against nE300) DEK-fragments. After incubation for 15 min at room temperature, the samples were centrifuged for 15 min at 14 000 r.p.m. The pellets were dissolved in 1% SDS and deproteinized with Proteinase K for 30 min at 55°C. The DNA was precipitated by ethanol, dried and finally resolved in loading buffer. Final analysis was carried out by agarose gel electrophoresis (0.8%, 17 h, 0.5× TBE, 2 V/cm) and SybrGold staining (MobiTec).

DNA pull-down assay. (A) Sephadex protein A beads were coupled with 6 μg of either unspecific rabbit IgGs or rabbit monospecific DEK antibodies for 1 h at 4°C, followed by the addition of 200 ng of the recombinant SAF-box fragment 137–183. After immobilization, supernatants (S) and the SDS-extracted beads (P) were analyzed by SDS–PAGE (18%) and immunoblotting with DEK specific antibodies. A molecular weight marker is indicated in kDa. (B) Binding of immobilized SAF-box peptides to DNA. Sephadex beads coated with the SAF-box peptide 137–183 prepared exactly as in (A) were incubated with a radiolabeled equimolar mixture of MAR (MII) and non-MAR DNA (pUC18) in the absence (IgG; α-DEK) or presence of increasing amounts of unspecific competitor DNA (+competitor DNA). Bound DNA was eluted from the beads and analyzed on 0.6% agarose gels followed by autoradiography. The relative intensities of both DNA fragments were determined by a densitometry program (NIH-Imager) and are shown in the lower panel of the graph. (C) An aliquot of 600 ng of full-length his-DEK was incubated with a radiolabeled equimolar mixture of MAR (MII) and non-MAR DNA (pUC18) in the absence or presence of increasing amounts of unspecific competitor DNA. The samples were centrifuged and the pellets were analyzed by agarose gel electrophoresis followed by autoradiography. The relative intensities of the both DNA forms were analyzed like in (B) and shown the lower panel.

In conclusion, we show that the isolated SAF-box peptide of the DEK protein possesses an intrinsic DNA-binding activity that could, however, not be detected in solution, but becameapparent after the peptides were immobilized and denselypacked on a Sephadex-bead matrix. A central peptide (amino acids 87–187) that includes the SAF box plus ∼50 N-terminal residues binds as efficiently as the full-length DEK to DNA. Therefore, the sequence preceding the SAF box appears to induce an interaction between single peptides creating a platform that favors a ‘mass binding mode’. Indeed, when incubated with DNA, the central peptide formed large complexes that remained in the slots of the agarose gel exactly as the full-length DEK does (Figure 2B). We investigated whether the central peptide shares other properties with intact DEK.

The central peptide binds to four-way junction DNA

We have recently shown that the full-length DEK preferentially binds to four-way junction DNA (13), and we investigate here whether the central peptide and other selected DEK-derived peptides share this DNA-binding mode. The DNA substrates were a set of labeled oligonucleotides that could be annealed to form eitherlinear or four-way junction DNA (Figure 4A). The full-length DEK as well as the DEK-derived protein fragments bound readily to both DNA forms as detected by the mobilityshifts of the resulting protein–DNA complexes (Figure 4B and C, lanes 2 and 7). Their preference for four-way junction DNA was assayed in competition experiments using unlabeled linear DNA as competitor. As expected, the binding to labeled linear DNA could be easilysuppressed by competing with unlabeled linear DNA (Figure 4B and C, lanes 8–10), but the binding to four-way junction DNA remained largelyresistant to competing straight DNA in cases when the full-length DEK (Figure 4B, lanes 3–5) and peptides including the sequence between residues 87 and 187 were used as binding partners (Figure 4C, 1–187, 87–187, lanes 3–5, respectively). Peptides that lacked the central DNA-binding domain but contained the C-terminal DNA-binding domain (Figure 1) bound efficiently to DNA (Figure 4C, 187–375 and Δ87–187), but showed no preference for four-way junction DNA, since competing linear DNA suppressed the binding to both labeled linear and labeled four-way junction DNA (Figure 4C, 187–375 and Δ87–187).

Figure 4

Binding of DEK and DEK-derived peptides to four-way junction (4WJ) and straight DNA. (A) Duplex (asterisk) and four-way junction (4WJ) DNA (double asterisk), for molecular sizes see (13). Oligonucleotides 1–4 are partially complementary to each other and assemble to the indicated four-way junction DNA upon annealing. (B) Full-length his-tagged DEK in bandshift assays. Oligonucleotides were annealed, purified on native polyacrylamid gels and then labeled. Labeled 4WJ DNA (lanes 1–5) and labeled duplex DNA (lanes 6–10) was incubated without (lanes 1 and 6) or with recombinant his-DEK (at molar ratio of DEK/DNA 50 for 4WJ and 100 for duplex DNA) without (lane +DEK) or in the presence of increasing amounts of unspecific, unlabeled duplex competitor DNA (at molar ratios: 10, 100 and 500) for 1 h at 37°C. After separation on native 8% polyacrylamid gels, the bands were visualized by autoradiography. (C) Preferential binding of SAF box carrying fragments to 4WJ DNA. Bandshifts were performed as in (B) (molar ratios for all investigated peptides DEK/DNA: 50 for 4WJ DNA, lanes 2–5; 100 for duplex DNA, lanes 7–10 or 0; lane 1, −DEK). Unspecific duplex competitor DNA was present in molar ratios 10, 100 and 500 or absent (lane +DEK). The used DEK derived fragments are indicated above the figures (1–187, 87–187, 187–375 and Δ87–187) and are schematically represented in (D).

We thusconclude that it is the central DNA-binding domain, including the SAF box, which is responsible for the specificity of DEK for non-linear four-way junction DNA.

The SAF box containing fragment induces negative supercoils

The full-length DEK protein changes the topology of closed circular DNA by introducing positive supercoils (12). As shown previously (14) and above in Figure 2C, the central fragment is also able to induce topological changes in closed circular DNA. However, the directionality of the supercoils introduced by this fragment has not been determined. To investigate this point, we first incubated DEK-derived peptides with closed circular SV40 DNA in the presence of type I DNA topoisomerase. The DNA samples were analyzed after deproteinization by 2D gel electrophoresis using chloroquine in the second dimension.

We confirmed that the full-length DEK induced positive superhelical turns, but were surprised to find that the central peptide (amino acids 87–187) induced negative supercoils (Figure 5A). We investigated a number of other DEK-derived peptides and found that sequences on both, the N-terminal side and on the C-terminal side of the central peptide, are necessary for the formation of positive supercoils. The smallestunit that we have found to be capable of inducing positive supercoils includes the amino acids 68–226. Deletions of 18–20 amino acids on either the C-terminal or the N-terminal side of this unit produced peptides that changed the topology of DNA by introducing negative turns.

Figure 5

Changing the topology of relaxed circular DNA. (A) The 2D gel electrophoresis. A standard topology assay was performed without protein (−DEK) or with recombinant full-length his-DEK (+DEK), 1–178, 87–187 or 68–226 at molar ratios of 150 (DEK/DNA). The purified DNA was separated by standard agarose gel electrophoresis for the first dimension (16 h, 2 V/cm) (from top to bottom). The gel was rotated by 90°, incubated in 0.25 μg/μl chloroquin (in 0.5× TBE) followed by a run for the second dimension (3 h, 4 V/cm) (from left to right). DNA was visualized by SybrGold staining. Direction (−; +) and number of introduces supercoils are indicated in the figures. (B) Schematic overview of all DEK-derived peptides tested in 2D gel electrophoresis. The direction of introduced supercoils is indicated for each peptide. Sequences 68–87 and 187–208 are necessary for the introduction of positive supercoils.

Thus, a central peptide including the SAF box can change the topology of closed circular DNA much like the full-length DEK does, but the direction of supercoiling is negative rather than positive as in the case of intact DEK. The reasonseems to be that the central peptide lacks adjacent sequences which are somehowinvolved in changing the sense of supercoiling from negative to positive.

The data of Figure 5 lead to the prediction that the 68–226 fragment, but not the 1–187 fragment, may reduce the negative superhelicity of minichromosomal SV40 DNA with its 25–28 constrained supercoils. This was indeed the case, and the data of Figure 6A show that the 68–226 fragment [like the full-length DEK, Figure 6A; DEK and see (12)] changes the topology of superhelical SV40 DNA, whereas the 1–187 fragment did not.

Figure 6

The binding of DEK-derived polypeptides to superhelical DNA. (A) Topology assay using SV40 minichromosomes. Left panel, control DNA. Full-length DEK, the 1–187 fragment and the 68–226 fragment were incubated at molar ratios of 50, 100 and 150 protein/DNA for 1 h at 37°C in the presence of topoisomerase I as described previously (12). The DNA was purified and analyzed by agarose gel electrophoresis and SybrGold staining. (B) DNA precipitation assay. Partially relaxed SV40 DNA (lane input, 20 ng) was incubated with increasing amounts of the polypeptides as indicated. After incubation for 15 min at room temperature, the protein–DNA complexes were precipitated. The DNA was purified and analyzed by agarose gel electrophoresis and SybrGold staining. (C) Densitometric analysis. The intensities of a selected DNA topoisomer with low superhelical density (filled square) and the intensities of highly supercoiled form I DNA (filled triangle) were determined by the densitometric program AIDA (Raytest). The results were plotted as relative amounts of precipitated DNA versus amounts of protein compared with input DNA.

The data could be explained by a different binding preference of the fragments tested. To investigate this point, we used the DNA precipitation assay with partially relaxed SV40 DNA as a binding substrate and increasing amounts of the 1–187 and the 68–226 fragments.

We detected that the 1–187 fragment bound preferentially to DNA topoisomers with reduced negative superhelicity, whereas the 68–226 fragment also bound to highly supercoiled DNA [like the full-length DEK, Figure 6B and C; see (13)]. Thus, the ability of the 68–226 fragment to associate with highly supercoiled DNA may at least partially explainwhy it reduces the negative superhelicity of SV40 DNA.

In the SAF-A protein, the SAF box appears to be the only DNA-binding motif. It directs the protein to the scaffold/matrix attachment regions (S/MAR) which are AT-rich stretches of DNA that associate with nuclear matrix preparations (26). In addition, SAF-A has a C-terminal RGG motif as an additional module, which is most likely involved in mRNA processing (27–30).

The SAF box of SpCCE clearly stabilizes the protein's interaction with four-way junction DNA, but is not its major DNA-binding determinant. However, SAF box is involved in DNA binding as demonstrated by specific amino acid exchanges that reduce the half-life of contacts between SpCCE and DNA. The crucial residues are two basic amino acids close to the second helix in the helix–loop–helix domain that constitute the SAF box (25) (Figure 1).

Like SpCCE and Ku70, DEK has two DNA-binding domains, namely a central domain including the SAF-box and a C-terminal domain of as yet undeterminedstructure (Figure 1A). However, it appears that the SAF-box domain is the main DNA-binding activity because a central fragment that includes the SAF box, but not the C-terminal binding domain, has DNA-binding properties that are similar to those of the entire DEK molecule. More precisely, DEK and the central domain prefer four-way junction over straight B-form DNA and induce supercoils into relaxed circular DNA. Peptides including the C-terminal domain bind well to DNA, but do not have the specificities of the full-length DEK or the central peptide. We assume that the second DNA-binding domain stabilizes the interaction of DEK with DNA and modulates its interaction in vivo and in vitro as it overlaps with major phosphorylation sites. Indeed, previous work has shown that a phosphorylation of DEK weakens DEK–DNA contacts (15).

Like SAF-A, DEK's isolated SAF box has a verylow affinity to DNA that cannot be determined by standard DNA-binding assays in solution (Figure 2B). Rather, it is necessary to fix many SAF boxes on a Sephadex matrix forming a DNA-binding platform that firmlyassociates with DNA. The combined binding activities of immobilized SAF boxes show a slight preference for AT-rich S/MAR sequences (Figure 3B). The full-length DEK protein has a similar preference (Figure 3C), but it is in any case much less pronounced than the affinity of SAF-A for S/MAR (17). It is therefore not clear whether DEK's preference for S/MAR is physiologicallysignificant. In addition, AT-rich sequences are known to assume unusual DNA structures (21,22) and it is possible that DEK recognizes these structures and not the sequence. We note though that one-tenth or so of all nuclear DEK fractionates with standard nuclear matrix preparations (31,32) (data not shown). However, the majority of nuclear DEK can be released from chromatin by milddigestion with micrococcal nuclease (8). It will be an interestingproblem for futureresearch to determine how DEK is distributed on chromatin, and how it is directed to its binding sites on chromatin. A possibility is that DEK associates with transcription factors at promoters or enhancers changing the path of the DNA to which it is bound, much like the better known HMGA or HMGBarchitectural proteins (33–36). Like DEK, these proteins are known to preferentially bind to four-way junction and superhelical DNA (36–38).

Anothersimilarity is that HMGA and HMGB proteins, such as DEK, induce supercoils into relaxed circular DNA. Whether this property has physiologicalconsequencesin vivo is not known. The topological changes are probablycaused by the extensivetwisting of DNA that occurs in the large DEK–DNA complexes which form in vitro (13). In any case, it is interesting to note that the central peptide (amino acids 87–187) introduces negative supercoils, whereas a slightly larger peptide (amino acids 68–226) induces positive supercoils (like the full-length DEK). This change in directionality cannot simply be explained by the known properties of DEK. Clearly, a determination of the 3D structure would help to successfullyaddress this issue.

In conclusion, we have shown here that the SAF box is the major DNA-binding domain of DEK because a central fragment of ∼100 amino acids that includes the SAF box has many of the DNA properties of the full-length DEK, while the C-terminal second DNA-binding domain appears to influence the overall protein–DNA contacts.

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References

The (6;9) chromosome translocation, associated with a specific subtype of acute nonlymphocytic leukemia, leads to aberrant transcription of a target gene on 9q34

M. von Lindern et. al

Mol. Cell. Biol., 1990

Autoantibodies to DEK oncoprotein in a patient with systemic lupus erythematosus and sarcoidosis

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